Conformationally abnormal forms of tau proteins and specific antibodies thereto

ABSTRACT

The invention relates to antibodies with a specificity to an abnormally truncated form of tau protein, which is conformationally different from normal tau, and does not bind to normal tau protein, conformationally different tau proteins (“tauons”) and diagnostic and therapeutical aspects in relation to Alzheimer&#39;s disease and related tauopathies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/470,928, filed on Nov. 25, 2003, which is a U.S. national phaseapplication under 35 U.S.C. § 371 of PCT Application No. PCT/EP02/00897filed 29 Jan. 2002, which claims priority to Austrian Application No. A175/2001 filed 2 Feb. 2001. The entire text of each of theabove-referenced disclosures is specifically incorporated by referenceherein without disclaimer.

BACKGROUND

The invention relates to Alzheimer's disease and to other tauopathies.Alzheimer's disease (AD) is the most common chronic neurodegenerativedisorder which is characterized clinically by a progressive andirreversible loss of cognitive and behavioral function. The disease canpersevere for over 10 years, advancing from mild symptoms to extremelysevere manifestations. AD afflicts approximately 10% of the populationover the age of 65 and 20% of the population over the age of 80. As aresult of growing of Western societies, the number of persons afflictedis rising: already there are five million sufferers in the USA alone andby the end of the year 2000, there will be roughly 18 million peoplewith dementia in the world. Of these, it is thought, that about twothird of cases, i.e. 12 million, will be Alzheimer's disease. It is thefourth largest killer in the Western world after the heart diseases,cancer and strokes. The number of people with dementia is risingquickly. By 2025, there will be twice the number of people with dementiain the developed world as there were in 1980. The cost to the societyfor looking after the sufferers is enormous. For example, the costs tothe US society for diagnosing and managing AD, primarily for custodialcare, is currently estimated at US$ 80 billion annually. Currently,neither presymptomatic diagnostic test nor cure for AD is available. Thedisease is therefore clinically diagnosed after appearance of symptomsprimarily by exclusion of other forms of dementia. Accumulation of theclassical hallmarks, senile (neuritic) plaques and neurofibrillarytangles (NFT) in the AD brains, observed 93 years ago by the Bavarianpsychiatrist Alois Alzheimer in 1907, still remain the neuropathologicalcharacteristic of AD.

The common denominator of intracellular neurofibrillary structures(neurofibrillary tangles, dystrophic neurites, and neuropil threads) arepaired helical filaments (PHFs). The major protein subunit of the PHFsis microtubule associated protein tau in abnormally hyperphosphorylatedform (Grundke-Iqbal et al., 1986; Wischik et al., 1988 a,b). Neuronswith neurofibrillary changes degenerate, and the degree of thisdegeneration directly correlates with the degree of dementia in theaffected individuals (Blessed et al., 1968). Normal tau is a microtubuleassociated protein that distributes mainly to axons. Tau protein istaking part in modulating the assembly, spatial organization andbehavior of microtubules (MT) in neurons and probably glial cell bodies(Drewes et al., 1998; Drubin and Kirschner, 1986; Lo-Presti et al.,1995). Tau proteins are encoded by a single gene located on chromosome17, but are detected as multiple isoforms in tissue extracts from adultbrains (Goedert et al., 1989; Himmler A., 1989; Kosik et al., 1989).Heterogeneity of tau proteins is in part due to alternative splicing,giving rise to six isoforms in the adult human brain. These distinctisoforms differ by the presence or absence of 29- or 58-amino acidinserts in the amino-terminal region and by the addition or deletion ofa tandem repeat (which can be repeated either 3 or 4 times) in acarboxy-terminal region of tau referred to as microtubule bindingdomain. This region is composed of imperfect repeats of 31 or 32 aminoacid residues. In humans, the smallest tau isoform contains 352 aminoacid residues with three tandem repeats in the MT-binding domain and noamino terminal inserts, whereas the largest isoform contains 441residues with four repeats and both amino terminal inserts. Forsimplicity, all numbering in this patent application refers to thelongest human tau protein isoform, htau40, containing all inserts (441amino acid long) according to Goedert et al. (1989).

A number of neurological diseases are known to have filamentous cellularinclusions containing microtubule associated protein tau e.g.Alzheimer's disease (AD), progressive supranuclear palsy (PSP),corticobasal degeneration (CBD), Pick's disease (PiD) and a group ofrelated disorders collectively termed frontotemporal dementia withParkinsonism linked to chromosome 17 (FTDP-17), amyotropic lateralsclerosis (ALS), Creutzfeldt-Jakob disease (CJD), dementia pugilistica(DP), Gerstmann-Straussler-Scheinker disease (GSSD), Lewy body diseaseand Huntington disease (Dickinson et al., 1998; DiFiglia et al., 1997;Forno, 1986; Hirano and Zimmerman, 1962; Nishimura et al., 1995;Prusiner 1996; Reed et al., 1998; Roberts, 1998; Schmidt et al., 1996;Shankar et al., 1989; Spillantini et al., 1998). Although the etiology,clinical symptoms, pathologic findings and the biochemical compositionof inclusions in these diseases are different, there is emergingevidence suggesting that the mechanisms involved in aggregation ofnormal cellular proteins to form various filamentous inclusions arecomparable. It is believed, that an initial alteration in conformationof microtubule associated protein tau, that initiates generation ofnuclei or seeds for filament assembly, is a key feature. This processcan be influenced by the posttranslational modification of normalproteins, by mutation or deletion of certain genes and by factors thatbind normal proteins and thus alter their conformation. The tau proteinis very hydrophilic. It can be readily extracted from brain tissue orcultured cells. In comparison, filamentous tau extracted fromAlzheimer's diseased brain tissues is relatively insoluble. Besidesphosphorylation, insoluble and normal soluble tau differ in the extentof posttranslational modifications, which include glycosylation,glycation, ubiquitination and racemization (Kenessey et al., 1995; Ko etal., 1999; Mori et al., 1987; Wang et al., 1996; Yan et al., 1994). Themechanism by which tau protein is modified to take part in filamentformation in AD is unknown. Tau is one of the most soluble proteinsknown (Cleveland 1977 a,b; Lee et al. 1988) and therefore itsaggregation in AD is particularly enigmatic. Phosphorylation of tauaffects the potential of tau to form aggregates, producing eitherstimulatory or inhibitory effects, presumably depending on the site ofphosphorylation (Crowther et al., 1994; Schneider et al., 1999). Many invitro studies demonstrate that in the presence of the reducing agent,dithiothreitol (DTT), unsaturated free fatty acids, RNA orglycosaminoglycans, normal tau can be transformed into filaments(Goedert et al., 1996; Kampers et al., 1996; Perez et al., 1996; Wilsonand Binder, 1997). Furthermore, the process of filament formation canalso be accelerated by the presence of cross-linked tau generatedthrough oxidation at Cys322 (Schweers et al., 1995). The parameters thathave been varied in different filament assembly studies have includedtau protein concentration, pH, and ionic strength of the incubation ismany fold higher than exists in the cytoplasm under physiologicalconditions. Examination of in vitro formed tau filaments by scanningtransmission electron microscopy (STEM) showed that these filamentsdiffer from native paired helical filaments (Ksiezak-Reding, 1998). Inthe absence of glycans or RNA, no PHF-like filaments are detectable insamples containing unphosphorylated or phosphorylated wild type tau;normal tau. Studies of chemically cross-linked, heparin treated tauindicate that heparin treatment induces conformational change in tauprotein (Paudel and Li, 1999). Taken together the in vitro data suggest(a) that the microtubule binding domain is important for assembly of taufilaments; and (b) that formation of tau filaments requiresconformational change(s) of tau. Simultaneously these studies show thatnone of tau modifications described, are alone capable to inducefilamentous tau formations that correlate with clinical expression ofAlzheimer's disease. Identification and description of factors importantfor the initiation of tau changes leading to filament formation indisease conditions would be important for the development ofpresymptomatic diagnostic markers and therapeutic agents to interferethe progression of tauopathies.

SUMMARY OF THE INVENTION

An object of the present invention is therefore to provide a reliabledrug target for early therapeutic intervention in Alzheimer's diseaseand other tauopathies. Furthermore it is desired to provide a specificmonoclonal antibody capable of specific detection and interaction withthis drug target. This antibody should not only be suitable forpresymtomatic detection of the molecule but for inhibition andelimination of this molecule as well, hence being suitable forpresymtomatic diagnosis, treatment and prevention of Alzheimer's diseaseand other tauopathies.

These objects are addressed with the present invention which relates inone aspect to an antibody with a specificity to abnormal forms of tauprotein which are conformationally different from normal tau, saidantibody being non-specific for a normal tau protein. Such abnormalforms of tau proteins represent a novel family of molecules, intra- andextra-neuronally located soluble and insoluble, preferably abnormallytruncated, forms of tau proteins, which are conformationally differentfrom normal tau (Novak et al., 1991, 1993). It could be shown with thepresent invention that these conformationally different forms of tauproteins—which are called “tauons” within the present specification—areseeds, nucleation centers in a self-propagating process of filamentoustau formations that is correlative to clinical expression of Alzheimer'sdisease thus tauons are important therapeutic targets for Alzheimer'sdisease. The tauons according to the present invention may be abnormallytruncated tau proteins. Biological activity of tauons can be inhibitedin vitro and inside of neurons by the antibodies according to thepresent invention. These antibodies have a capacity to stain presence oftauons in presymtomatic stages I, II and III of AD, which makes themsuitable for presymtomatic diagnosis of this disease. It is critical forthe antibodies according to the present invention that only theconformationally different form of tau protein (i.e. the “tauon”) isrecognized by this antibody whereas the normal tau protein does not bindto the antibodies according to the present invention.

Within the course of the present invention AD truncated forms ofmicrotubule associated protein tau were purified to homogenity and shownto be a major part of filamentous tau isolations from Alzheimer'sdiseased neurons. The amino acid sequence data indicated that thebackbone of tauons is indistinguishable from that of protein tau buttauons could be distinguished immunologically from normal human tau bythe different conformation as revealed by the conformation specificmonoclonal antibodies according to the present invention. Specificexamples for such antibodies are the monoclonal antibody DC-11 which isproduced by the hybridoma cell line which was deposited in the EuropeanCollection of Cell Cultures (ECACC) under deposit No. 00082216 andmonoclonal antibody DC-11/I which is produced by the hybridoma cell lineDC-1/I and was deposited in the ECACC under deposit No. 00082215. Thisfamily of monoclonal antibodies which is provided with the presentinvention is defined by recognition of tauon-specific conformationwithout recognizing normal human soluble tau. The different conformationcompared to normal human tau, was attributed pathologically to abnormaltruncation at the N-terminus or at the C-terminus or at both termini oftau molecule in the samples tested so far from Alzheimer's diseasepatients. Interestingly, the different conformation was regardless oftau isoform and level of phosphorylation. The indispensable pathologicalrequirements for tauons to attain typical conformation is presence ofprolin rich and microtubule binding domains and truncated flankingregion(s). Furthermore tauons could be distinguished from normal humantau by their pathological activities, namely that tauons represent aseed, nucleation center, that initiates tau aggregation and tauonsdisassemble microtubules assembled from normal tau and tubulin. Tauonspreincubated with antibodies according to the present invention,especially monoclonal antibodies of the DC-11 family, showed nodiassemble capacity or assembled microtubules from normal tau andtubulin. Moreover, tauons cause upon microinjection to differentiatedhuman neurons significant displacement of endogenous tau frommicrotubule bound tau fraction, retraction of neuronal processes anddegeneration of the cells. If tauons are microinjected together withmonoclonal antibodies according to the present invention, noneurodegenerative changes were observed in differentiated neurons. Thisshows that the antibodies according to the present invention, especiallythe DC-11 monoclonal antibodies, inhibit tauons activity intraneuronallyand therefore could be used as intracellular drugs (for example astherapeutic intracellular antibodies, intrabodies).Immunohistologically, as seen with the antibodies according to thepresent invention, tauons occur already in presymptomatic stages I, IIand III in pre-α-neurons, in both the transenthorinal and enthorinalregion of AD, therefore, after proper coupling of tracers, antibodiesaccording to the present invention could be used for intravitalpresymptomatic diagnosis for AD.

Preferably, the antibody according to the present invention exhibits aspecificity of at least 50%, preferably at least 90% to theconformationally different form of tau (“tauon”) compared to theantibody DC-11. Specificity may be tested by any standard test availablefor detecting antibody's specificity, e.g. ELISA tests,radioimmuno-assays, atomic force microscopy with cantilever-boundbinding partners, etc.

Generally, all antibodies which are specifically reactive with theconformationally different tau protein, especially abnormally truncatedforms thereof, but not with normal soluble tau are also included withinthe scope of the present invention.

Preferably, the antibody according to the present invention is said tobe “specifically reactive” with a molecule if it is capable of bindingwith a molecule to thereby couple the molecule to the antibody. The term“epitope” is meant to refer to that portion of an antigen which can berecognized and bound by an antibody. An antigen may have one or morethan one epitope. An “antigen” is capable of inducing an animal toproduce antibody capable of binding to an epitope of that antigen. Thespecific reaction referred to above is meant to indicate that theantigen will immunoreact, in a highly selective manner, with itscorresponding antibody and not with the multitude of other antibodieswhich may be evoked by other antigens.

Especially preferred antibodies according to the invention are derivedfrom deposited hybridoma cell lines DC-11 (ECACC deposit No. 00082216)and DC-11/I (ECACC deposit No. 00082215) exhibit high specificity andselectivity and will react with conformationally different form of tau(“tauon”), but not with normal soluble tau. Specificity may be tested byany standard test available for detecting antibody's specificity, e.g.ELISA tests, radioimmuno-assays, etc.

“Antibody” as used herein is meant to include intact molecules andfragments thereof as well as synthetic and biological derivativesthereof, such as for example Fab, F(ab′)₂ an F_(V) fragments-free orexpressed e.g. on the surface of filamentous phage on pIII or pVIII orother surface proteins, or on the surface of bacteria, which are capableof binding an antigen. Fab, F(ab′)₂ and F_(V) fragments lack the F_(C)fragments of intact antibody, clear more rapidly from the circulationand may have less non-specific tissue binding of antibody. FurthermoreF_(V) antibody (often called as minibody) can be easily engineered tocarry on its C-terminus specific tracer and used for early intravitalpresymtomatic diagnosis of AD, since stage I, II and III of AD that isrecognized by the antibodies according to the present invention is notassociated with intellectual decline.

Within the present invention, monoclonal antibodies or monoclonalantibody fragments are preferred. Therefore, according to another aspectthe present invention also relates to hybridoma cell lines producing amonoclonal antibody according to the present invention.

The term “tau” as used in the present application refers to the longestisoform of human microtubule associated protein tau containing allalternatively spliced inserts as described in M. Goedert et al., 1989.

According to another aspect of the present application the inventionrelates to an abnormally truncated form of tau protein which is aconformationally different form of tau protein, said conformationallydifferent form of tau protein specifically recognizable by an antibodyaccording to the present invention.

Accordingly, the present invention is drawn to a novel family ofmolecules intra- and extraneuronally located soluble and insolubleabnormally truncated form of tau proteins which are conformationallydifferent from normal tau and are called “tauons”.

“Tauons” therefore are conformationally different forms of tau proteinwhich are specifically recognized by the antibodies according to thepresent invention. Tauons useful in the present invention comprise thesequence according to SEQ ID NO. 1 and may be flanked by further aminoacids (see SEQ ID NO. 2 and SEQ ID NO: 3). The tauons conveniently arein the range from about 100 to 400 amino acids and represent truncatedforms of tau protein in this range. The tauons according to the presentinvention may be abnormally truncated at the N- or C-terminus or at bothtermini (see FIGS. 2-13). The term “abnormally truncated” as used hereinrefers to tau peptides (“tauons”) identified in diseased neurons in ADwith tauon specific monoclonal antibodies provided with the presentinvention.

Abnormally truncated forms of human tau proteins—tauons—can be preparedby using any of numerous well known synthetic recombinant techniques.Briefly, most of the techniques which are used to transform cells,construct vectors, extract messenger RNA, prepare cDNA libraries, andthe like are widely practiced in the art, and most practitioners arefamiliar with the standard resource materials which describe specificconditions and procedures. However, for convenience, the followingparagraphs may serve as a guideline.

The most commonly used prokaryote system for the production ofrecombinant proteins remains E. coli, however, other microbial strainsmay also be used, such as Bacilli, for example Bacillus subtilis,various species of Pseudomonas, or other bacterial strains. In suchprokaryotic systems, plasmid vectors which contain replication sites andcontrol sequences derived from a species compatible with the host areused. Commonly used prokaryotic control sequences include promoters fortranscription initiation, optionally with an operator, along withribosome binding site sequences.

A wide variety of eukaryotic hosts are also now available for productionof recombinant foreign proteins. As in bacteria, eukaryotic hosts may betransformed with expression systems which produce the desired proteindirectly, but more commonly, signal sequences are provided to effect thesecretion of the protein. Eukaryotic systems have the additionaladvantage that they are able to process introns which may occur in thegenomic sequences encoding proteins of higher organisms. Eucaryoticsystems also provide a variety of processing mechanisms which result in,for example, glycosylation, oxidation or derivatization of certain aminoacid residues, conformational control, and so forth.

Commonly used eukaryotic systems include yeast, insect cells, mammaliancells, avian cells, and cells of higher plants. The list is notexhaustive. Suitable promoters are available which are compatible andoperable for use in each of these host types as well as are terminationsequences and enhancers, as e.g., the baculovirus polyhedron promoter.As above, promoters can be either constitutive or inducible. Forexample, in mammalian system, the MTII promoter can be induced by theaddition of heavy metal ions.

The particulars for the construction of expression systems suitable fordesired host are known to those in the art. For recombinant productionof the protein, the DNA encoding it is suitable ligated into theexpression system of choice, and the system is then transformed into thecompatible host cell which is then cultured and maintained underconditions wherein expression of the foreign gene takes place. Thetauons of this invention produced this way, are recovered from theculture, either by lysing the cells or from the culture medium asappropriate and known to those in the art.

Correct ligations for plasmid construction can be confirmed by the firsttransforming a suitable host with the ligation mixture. Successfultransformants are selected by ampicillin, tetracycline or otherantibiotic resistance or using other markers depending on the mode ofplasmid construction, as is understood in the art.

The present invention therefore relates to a preparation of tauons,especially from human or recombinant resources, being essentially freeof other proteins, especially from normal tau proteins. Suchpreparations may be provided by procedures involving an immunoaffinitystep using the antibodies according to the present invention.Preferably, the preparation according to the present invention containsmore than 80% tauons, especially more than 95% tauons, of total protein.

Further the present invention also relates to a kit for detectingtauons, abnormally truncated forms of tau protein, which areconformationally different from normal tau in a sample of Alzheimer'sdisease brain tissue or in a sample of a body fluid comprising anantibody according to the present invention and a suitable container forproviding the probe. It is possible to provide the antibodies in a kitfor detecting or isolating of tauons. With the help of antibodiesaccording to the present invention tauon proteins may be detected andisolated from various sources including Alzheimer's diseased neurons oftransenthorinal, enthorinal region and hippocampus. Tauons isolated inthis way may be further used as immunogen for immunization e.g. of micefor construction of hybridomas producing specific monoclonal antibodiesagainst tauons not recognizing normal full length tau. This methodcomprises identifying and releasing neurons from transenthorinal,enthorinal and hippocampal region of Alzheimer's diseased brain tissuesinto the solution preserving abnormal conformation of tauons.

After preparation and purification, tauons are used as immunogens andinjected subcutaneously to mice in monthly intervals. Spleens from theseanimals are used for construction of hybridomas producing monoclonalantibodies against tauons. These can be produced using well-establishedhybridoma techniques first introduced by Köhler and Milstein (see M.Köhler and C. Milstein, “Continuous Cultures of Fused Cells SecretingAntibody of Pre-Defined Specificity”, Nature, 256, pp. 495-497, 1975).After a sufficient long immunization, antibody-producing lymphocytes areobtained from the animal either from the spleen, lymph nodes orperipheral blood. Preferably, the lymphocytes are obtained from thespleen. The splenic lymphocytes are then fused with a myeloma cell line,usually in the presence of a fusing agent such as polyethylene glycol(PEG). Any of number of myeloma cell lines may be used as a fusionpartner according to standard techniques; for example theP3-NS1/1-Ag4-1, P3-x63-Ag8.653 myeloma lines. The resulting cells, whichinclude the desired hybridomas, are then grown in a selective medium,such as HAT medium, in which unfused parental myeloma or lymphocyte celleventually die. Only the hybridoma cells survive and can be grown underlimiting conditions to obtain isolated clones. The supernatants of thehybridomas are screened for the presence of antibody of that desiredspecificity, e.g. by immunoassay techniques using the antigen that hadbeen used for immunization. Positive clones can then be subcloned underlimiting dilution condition or on soft agar and the monoclonal antibodyproduced can be isolated. Hybridomas produced according to these methodscan be propagated in vitro or in vivo (in ascites fluid) usingtechniques known in the art. Commonly used methods for purifyingmonoclonal antibodies include ammonium sulphated precipitation, ionexchange, chromatography, and affinity chromatography (see e.g., H. Zolaet al., “Techniques for the Production and Characterization ofMonoclonal Antibodies”, in Monoclonal Hybridoma Antibodies: Techniquesand Applications, J. G. R. Hurell (ed.), pp. 51-52 (CRC Press 1982)).

Preferably, the kit according to the present invention further containsmeans for detecting the binding event of said antibody binding to saidconformationally different tau protein. Preferably secondary antibodiesespecially secondary antibodies which are specifically labeled. Also themagnetic beads technology may be used within the scope of the presentinvention as well as other protein identifying methods using antibodies.The method comprises identifying in a test sample from the person,tauon, which is abnormally truncated tau protein. “Test sample” as usedherein refers to biological sample from the person that is suspected ofcontaining tauons. Test sample can comprise brain tissue havingabnormally truncated tau proteins, such as hippocampal tissue or frontalcortex tissue or, the test sample can comprise cerebrospinal fluid(CSF). In a preferred embodiment, the test sample comprises CSF and theprotein identified is CSF-tauon. Identification of abnormally truncatedtau proteins—tauons—conveniently comprises identifying in the testsample antigens capable of binding with antibodies specifically reactivewith abnormally truncated tau proteins—tauons—comprising the sequence(SEQ ID NO: 1) and flanked by amino acids such that said tauons are inrange from about 100 to 400 amino acids in length and characterized bytauon specific conformation different from normal soluble protein tau,or antibodies specifically reactive with abnormally truncated tauproteins—tauons—comprising the sequence (SEQ ID NO: 1) and flanked byamino acids such that said tauons are in range from about 100 to 400amino acids in length and characterized by tauon specific conformationdifferent from normal soluble protein tau. The presence of a tauonindicates a disease associated with the accumulation of the tauons in ADpatients and other sufferers with tauopathies.

A further aspect of the present invention relates to a method fordetecting an abnormally truncated form of tau protein which isconformationally different from normal tau in a body fluid of a patientcomprising mixing said body fluid with an antibody according to thepresent invention, detecting the presence of a binding event between theantibody and the conformationally different tau protein (tauon) andoptionally measuring the amount of conformationally different tauprotein being bound to said antibody. The presence of a tauon indicatesa disease associated with the accumulation of the tauons in a personincluding AD and other tauopathies. The body fluid of a patient may beany biological test sample from a person that is suspected of containingtauons. This body fluid can comprise brain tissue such as hippocampaltissue or frontal or a cortex tissue or cerebrospinal fluid (CSF). In apreferred embodiment the body fluid comprises CSF and the proteinidentified is CSF-tauons.

This identification of tauons can conveniently be accomplished bybiochemical or cytochemical means or by enzyme immunoassays such asdescribed in many manuals of immunoassay producers as it is understoodin the art. When biochemical means are used preferably 0.01 to 10 g,especially 0.5 to 1 g, of tissue containing diseased tau protein isused, run on a gel and identified by Western blot. Such a technique isbelieved do be adequate in the absence of age matched controls whichhave been shown to be non-reactive with the antibodies according to thepresent invention. Cytochemical means, staining, has shown no reactivitywith normal tissue.

CSF from patients with AD and patients with non-AD neurological diseasesas well as normal subjects were surveyed by ELISA to quantitate level oftauons. The CSF tauon level was significantly increased in AD patientsas compared with that with patients with non AD neurological diseasesand controls. In AD, the significant increase was found irrespective ofage of onset, apolipoprotein E genotype and clinical stage. Westernblots of AD CSF proteins reveal several immunoreactive bands withapparent molecular weight between 50 and 15 kD consistent withabnormally truncated tau proteins. These results indicate thatCSF-tauons reflect that progressive accumulation of diseased tau causedby progression of AD.

According to a further aspect the antibodies according to the presentinvention may be used for the preparation of drug for the treatment ofAlzheimer's disease patients. The antibodies may be biotechnologicallymodified into single chain molecules equipped with targeting sequenceable to deliver them into the neuroblastoma cells expressing tauons.Inside of the present AD cellular model, antibodies bind tauons andinterfere with their pathological effects (sequestration of normal tau)and increase the degradation of abnormally truncated forms of tauprotein. In vitro assays (sequestration of tau protein, filamentassembly, microtubule disassembly) with abnormally truncated tauproteins and their correlation with severity of Alzheimer's disease showthat they are important drug targets.

The present invention will be described in more detail by the way of thefollowing examples and the figures to which the invention should not belimited.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an overview of the tauon preparation.

FIG. 2 shows a summary schematic representation of tauon amino acidsequences.

FIG. 3 shows minimal tauon.

FIG. 4 shows C-terminally truncated tauon.

FIG. 5 shows N-terminally truncated tauon.

FIG. 6 shows a schematic tau representation.

FIG. 7 shows human tau 37.

FIG. 8 shows human tau 39.

FIG. 9 shows human tau 40.

FIG. 10 shows human tau 43.

FIG. 11 shows human tau 44.

FIG. 12 shows human tau 46.

FIG. 13A and FIG. 13B show rat big tau.

DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS Example 1 Preparation of theMonoclonal Antibodies of DC 11 Family Specific for Tauons Preparation ofSoluble and Insoluble Tauons as Antigens for Immunization (FIG. 1)

For isolation of tauons from human AD brains, a new approach wasdeveloped which is partially based on the methods described by Kopke etal., (1993) and Greenberg and Davies (1990). Human brains, showingchanges characteristic for I.-III. Braak's stage of AD with short postmortem delay (PMD) were selected. Blocks of the temporal lobe includingthe enthorinal and transenthorinal regions, amygdala and hippocampalregion were selected. The tissue was dissected and immediately immersedinto minimal essential medium (Gibco). Tissue was finely minced andpushed through a 150 μm mesh wire screen. At this stage the brain samplewas divided into two aliquots: sample A and sample B.

Sample A was further processed in 20 mM TRIS, pH 8, 0.32 M sucrose, 10mM mercaptoethanol, 5 mM EGTA, 1 mM EDTA, 5 mM MgSO₄, 5 mM benzamidine,10 mM glycerolphosphate, 6 mM phenylmethylsulfonyl fluoride, 50 mMsodium fluoride, 5 μg/ml leupeptin, 1.5 μg/ml pepstatin and 2 μg/ml ofaprotinin and centrifuged at 25 OOO×g for 35 min at 4° C. to removecellular debris. The supernatant was then pelleted at 200 000×g for 40min. The resulting pellet was extracted with 8 M urea at roomtemperature for 70 min and spun at 300 000×g for 45 min at roomtemperature. The supernatant was dialysed for 24 hrs against the 10 mMTRIS pH 7.6 with frequent changes and then dialysed for 24 hrs against100 mM MES, 0.5 mM MgCl₂, 1 mM EDTA, 2 mM EGTA, 1 mM dithiotreithol,0.75 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride and 50 mM NaF, pH2.7. The precipitated proteins were removed by centrifugation at 200000×g for 40 min. The 200 000×g supernatant was dialyzed against 25 mMMES, pH 6.4, 0.5 mM MgCl₂, 0.1 mM EDTA and 1 mM dithiotreithol andsubsequently fractionated on Cellulose Phosphate column which wasequilibrated with the same buffer. The column was loaded with 2 mg/ml ofproteins and eluted with 20 ml of linear gradient of NaCl (0-1M) inequilibrating buffer. The proteins eluted with 0.1-0.8 M NaCl wereevaluated by Western blotting and concentrated by speed vacuumapparatus.

Sample B was put into 10 volumes of cold buffer (10 mM TRIS, 1 mM EGTA,0.8 M NaCl, 10% sucrose, pH 7.4) in a glass homogenizer. Aftercentrifugation at 27 000×g for 30 min at 4° C., the supernatant wassaved and the pellet was homogenized with the buffer and centrifuged at27 000×g for 30 min. The 27 000×g supernatants from both centrifugationswere combined, adjusted to 1% (wt/vol) N-lauroylsarcosine and 1%(vol/vol) β-mercaptoethanol and incubated at 37° C. for 3 hrs on shaker.After centrifugation at 35 000 rpm for 30 min, the pellet washomogenized in 5 ml of homogenizing buffer supplemented with 1%mercaptoethanol and filtered through 0.45 μm filter. The filtrate wascentrifuged at 35 000 rpm for 1 hr. The pellet was resuspended in 50 mMTris, pH 6.8 and extracted with 2.5% formic acid for 2 min and thencentrifuged at 10 000×g for 10 min to pellet insoluble material. Thesupernatant was dialysed overnight at 4° C. against 10 mM Tris, pH 7.4and centrifuged as previously. The resulting supernatant (fraction II)was concentrated using speed vacuum apparatus and evaluated by SDS-PAGEfollowed by Western blotting. Pellet from the sample B after extractionwith 2.5% formic acid containing insoluble tauons (fraction III) wassaved and used for immunizations and dot assay. Tauons from fractions(I, II and III) were pooled and used as antigens (see FIG. 1) forimmunization of mice.

Preparation of Hybridomas Producing Family of DC-11 MonoclonalAntibodies

Six weeks old Balb/c mice were divided into three groups, (A,B,C). Thefirst two groups (A,B) were primed with 50 μg of antigen in completeFreund's adjuvant (Sigma) and boosted five times at three weeksintervals with 50 μg of the same antigen (Ag) in incomplete Freund'sadjuvant. In the group A all doses were injected into foot pad and inthe group B doses of Ag were administered subcutaneously. The thirdgroup of mice was injected only with one dose directly into the spleenin PBS (intrasplenic immunization) and one week after such priming werespleens used for fusion. Three days before the fusion, mice in group Aand B were injected intravenously with 50 μg of immunogen in PBS. Spleencells from immunized mice were fused with NS/0 myeloma cells accordingto the method of Kontsekova et al., 1988. 10⁸ splenocytes were mixedwith 2×10⁷ NS/0 myeloma cells (ratio 5:1) and fused for 1 minute in 1 mlof 50% PEG 1550 (Serva) in serum free Dulbecco's modified Eagle's medium(DMEM) supplemented with 10% dimethyl sulphoxide. The fused cells wereresuspended in DMEM containing 20% horse serum, L glutamine (2 mM),hypoxanthine (0.1 mM), aminopterin (0.004 mM), thymidine (0.016 mM) andgentamycin (40 U/ml) at a density of 2.5×10⁵ spleen cells per well on96-well plates. Cells were incubated 10 days at 37° C. and growinghybridomas were screened for the production of anti-tauon specificmonoclonal antibodies by ELISA and by immunohistochemistry.

Anti Tauons Antibody Screening by ELISA

ELISA was used to detect monoclonal antibodies in hybridoma culturesupernatants which were directed against tauons. As a solid phase tauonsprepared as described above with a following modification were used.Pool of the speed vacuum concentrated tau from fractions was separatedby electrophoresis on polyacrylamide gel and truncated forms of tauprotein were recovered by electroelution according to the method byDonofrio et al., (1986) and evaluated by SDS-PAGE. Microtiter plateswere coated overnight with abnormally truncated tau proteins (10 μg/ml,50 μl/well) at 4° C. in PBS. After blocking with 1% nonfat dried milk toreduce nonspecific binding, the plates were washed with PBS-0.05% Tween20 and incubated with 50 μl/well of culture supernatant for 1 hr at 37°C. Bound monoclonal antibodies were detected with sheep anti-mouse Igconjugated with horse radish peroxidase (DAKO). The reaction wasdeveloped with o-phenylenediamine solution as a peroxidase substrate andstopped with 50 μl 2 M H₂S0₄. Absorbance at 492 nm was measured using aMultiscan MCC/340 ELISA reader (Labsystems). Readings at least twice ofthe value of the negative controls were considered positive.

Positive cultures were further subcloned in soft agar according to theprocedure of Kontsekova et al., 1991. Isolated subclones werere-screened for the production of specific anti-tauon monoclonalantibodies.

Immunohistochemical Screening of Anti Tauon Antibodies

Monoclonal antibodies identified as positive in anti-tauon ELISA andnegative on normal tau, were re-screened on AD brain tissues for theirspecificity as follows:

The brains of patients with AD removed at autopsy were sectioned at 1 cmintervals in the coronal plate and stored at −20° C. The blocks ofhippocampus, enthorinal, temporal, frontal, occipital and parietalcortex were fixed in 4% buffered paraformaldehyde at 4° C. for more than4 days. Series of frontal sections (50 μm) were cut on vibratome andstored in PBS (pH 7.0) at 4° C. Free floating vibratome sections werepretreated for 2-3 min with 98% cold formic acid, incubated withpre-immune serum in PBS/Triton X 100. The serum used was from the sameanimal species as that of secondary antibody. Incubation of sections wasdone with the monoclonal antibody positive in ELISA (as described above)for 60 min at 37° C.

Incubation with the second biotinylated antibody (Vectastain Elite kit,Vector) was performed for 1 hr at room temperature. Immunoreactions werevisualised with the avidin-biotin-peroxidase complex (Vectastain Elitekit, Vector) and 6 mg 3-3-diaminobenzidine-4 HCl (SIGMA), 250 mg NiCl₂(MERCK) in 10 ml 0.1 M acetate buffer (pH 6) with 100 μl H₂O₂. Reactionwas terminated by washing the sections in PBS/Triton (Kiss et al., 1988;Cuello et al., 1993, Thorpe and Kerr, 1994)

Example 2 Quantitative Determination of the Abnormally Truncated TauProteins (Tauons) Using Family of DC-11 Monoclonal Antibodies

Tauons were isolated as described above. The combination of monoclonalantibody DC 30 (recognizing both normal and pathological tau) and familyof DC-11 monoclonal antibodies (specific for abnormally truncated tau)allows quantification of tauons in the tested samples prepared fromAD-brains. Antibodies were purified from serum-free medium by protein Acolumn chromatography. The wells of high-binding microtiter plate (Nunc)were coated with mixture of DC-11 monoclonal antibodies at aconcentration of 10 μg/ml (50 μl/well) in PBS overnight at 4° C.Non-specific binding in wells was saturated by adding 200 μl of 1%nonfat dried milk in phosphate buffered saline (PBS) for 60 min at roomtemperature. The plates were washed 3 times with PBS-0.05% Tween 20(v/v). The serially diluted standards containing recombinant tauons atconcentrations ranging between 100-1000 pg/ml in PBS were added, as wellas the test samples containing AD-tauons in amount of 50 μl. Afterincubation for 60 min at 37° C., plates were washed and the horse radishperoxidase conjugated antibody DC 30 diluted 1/5000 in PBS was added (50μl/well) for 60 min at 37° C. After a final washing, 50 μl oforthophenylendiamine solution and 0.003% H₂O₂ were added to wells andplates were incubated in dark for 20 min. The reaction was stopped with50 μl of 2 M H₂SO₄. Absorbance at 492 nm was read in ELISA readerMultiscan MC344 (Labsystems, Finland).

The standard curve for recombinant tauons was constructed from theobtained values and the corresponding concentrations of tauons in testedsamples were determined from the standard curve.

Example 3 Detection of Tauons by Western Blotting Using MonoclonalAntibody DC-11

Purified recombinant full-length human tau and abnormally truncated tauproteins—tauons, were loaded on 5-20% gradient SDS-polyacrylamide gelsand run under denaturated conditions according to Laemmli (1970). AfterSDS-PAGE, the transfer on polyvinyl difluoride membrane (Milipore) wascarried out in 10 mM CAPS buffer pH 12 for 1 hr at 350 mA with cooling.After blotting, the membrane was washed in PBS and blocked with 1% driednonfat milk in PBS for 1 hr at room temperature. Transferred proteinswere incubated overnight at 4° C. with monoclonal antibody DC-11. Afterwashing with PBS-0.05% Tween 20 (v/v), rat anti-mouse immunoglobulinlabeled with horse radish peroxidase was used at a dilution 1/1000 andincubated 1 hr at room temperature. The membrane was then washed fourtimes in PBS-Tween 20, developed with substrate solution (12 mg4-chloro-1-naphtol, 4 ml methanol, 16 ml PB, 0.03% v/v H₂O₂) and thereaction was stopped in H₂O. Results indicated, that the antibody DC-11recognized solely abnormally truncated tau—tauons, by contrast themonoclonal antibody DC 30 is pan tau antibody recognizing universallyall known tau isoforms from many species (human, monkey, cow, pig, rat,mouse), regardless of their state of postranslational modifications.

Example 4 Immunohistochemical Identification of Tauons

Monoclonal antibody DC-11 family are suitable for visualization oftauons in AD-brains in different types of the immunohistochemicalprocedures.

Light Microscopic Labeling

The brains of patients with AD removed at autopsy were sectioned at 1 cmintervals in the coronal plate and stored at −20° C. The blocks ofhippocampus, entorhinal, temporal, frontal, occipital and parietalcortex were fixed in 4% buffered paraformaldehyde at 4° C. for more than4 days. Series of frontal sections (50 μm) were cut on vibratome andstored in PBS (pH 7) at 4° C. Free floating vibratome sections werepretreated for 2 min with 98% cold formic acid, incubated withpre-immune serum in PBS/Triton X 100. The serum used was from the sameanimal species as that of secondary antibody. Incubation of sections wasdone with monoclonal antibody DC-11 for 60 min at 37° C. Incubation withthe second biotinylated antibody (Vectastain Elite kit, Vector) wasperformed for 1 hr at room temperature. Immunoreactions were visualizedwith the avidin-biotin-peroxidase complex (Vectastain Elite kit, Vector)and 6 mg 3-3-diaminobenzidine-4 HCl (SIGMA), 250 mg NiCl₂ (MERCK) in 10ml 0.1 M acetate buffer (pH 6) with 100 μl H₂O₂. Reaction was terminatedby washing the sections in PBS/Triton (Kiss et al., 1988; Cuello et al.,1993, Thorpe and Kerr, 1994).

Light Microscopic Double Labeling

Free floating vibratome sections were pretreated for 2-3 min with 98%cold formic acid, incubated with pre-immune serum in PBS/Triton X 100.The serum used was derived from the same animal species as that ofsecondary antibody. Sections were incubated with first peroxidaseconjugated monoclonal antibody DC-11 at a dilution 1:1000 in blockingsolution (5% horse serum, PBS, 0.1% Triton) for 60 min at 37° C.,developed in 0.06% DAB, 0.01% H₂O₂ in PBS (pH 7.2).

Reaction was terminated by washing the sections in PBS/Triton.Incubation of the same sections with second monoclonal antibody was donefor 60 min at 37° C. Incubation with the biotinylated antibody(Vectastain Elite kit, Vector) was performed for 1 hr at roomtemperature. The reaction was visualized with theavidin-biotin-peroxidase complex (Vectastain Elite kit, Vector) and0.06% 3-3-diaminobenzidine-4 HCl (SIGMA), Q.01% H₂O₂, 2.5% NiCl₂ (MERCK)in 0.1 M acetate buffer and was terminated by washing the sections in0.1 M acetate buffer (Kiss et al., 1988; Cuello, 1993)

Counterstaining with Fast Cresyl Violet

After finishing immunohistochemical staining, the sections were placedon glass slides and put into thermostat for 60 min at 56° C. Afterincubation slides were immersed in distilled water for 5 min, stained infast cresyl violet solution for 5-10 min at 4° C., rinsed in water, andtransferred 25 to 96% ethanol until most of the cresyl violet stainingwas removed, cleared in xylen, and mounted with Entellan.

Immunofluorescent Staining

Free floating vibratome sections (30 μm) were pretreated for 2 min with98% cold formic acid, incubated with pre-immune serum in PBS/Triton X100. Sections were incubated with first primary monoclonal antibodyDC-11 for 60 min at 37° C. and then incubated for 30 min with aFITC-conjugated goat anti mouse secondary antibody (Immunotech) diluted1:500 in PBS/Triton at room temperature according to standard methodsused in the field. After washing with PBS, sections were incubated withTRITC-conjugated primary antibody for 60 min at 37° C. and mounted in0.1% paraphenylendiamin/glycerol solution.

Example 5 Microtubule Assembly and Microtubule Binding Assays withTauons Tubulin Purification

Tubulin was isolated from fresh pig brains, obtained from the localslaughter house, by temperature dependent cycles of microtubulepolymerisation and depolymerisation, followed by phosphocellulose(Watman P11 phosphocellulose) ion-exchange chromatography (Valee, 1986).

Microtubule Assembly Assay

Purified tauons (5 mM) were mixed with precleared purified tubulin (10mM) and GTP (1 mM) in the assembly buffer (100 mM PIPES pH 6.9, 2 mMEGTA, 1 mM MgSO₄) at +4° C. This tubulin concentration is below thecritical concentration for spontaneous assembly (Black, 1987). Sampleswere pipetted into quartz cuvettes preheated to 37° C. The change ofturbidity was measured spectrofotometrically in thermostaticallycontrolled spectrophotometer (LKB) and recorded as change of OD₃₅₀ in 10s intervals for a period of 30 min.

Microtubule Binding Assay

Binding curves of tauons with microtubules were measured as describedpreviously (Gustke, 1992). Purified tubulin was incubated at 37° C. inthe presence of GTP (1 mM) and taxol (20 μM) in the binding buffer (100mM PIPES pH 6.9, 1 mM EGTA, 1 mM MgSO₄, 1 mM DTT) for 10 min.Microtubules were stabilized by taxol which does not interfere with thebinding of tauons or normal tau protein and other MAPs, respectively(Valee, 1986; Wallis, 1993) thus eliminating the effect of microtubuleassembly. Tauons were added in concentrations 2.5 mM, 5 mM, 7.5 mM, 10mM, 15 mM, 20 mM, respectively. After centrifugation for 35 min, 43000×g, 37° C. the pellets were resuspended in P buffer (50 mM PIPES pH6.9, 1 mM EGTA, 0.2 mM MgCl₂, 5 mM DTT, 500 mM NaCl). Supernatant andpellets were analyzed on SDS-PAGE gels (Laemmli, 1970), stained withsilver (Bloom, 1987). The gels were scanned on HPScanJet(Hewlett-Packard) scanner and analysis performed on a Macintosh computerusing the public domain NIH Image program (developed at the U.S.National Institutes of Health and available on the Internet atrsb.info.nih.gov/nih-image/). The band intensities were converted toconcentrations using the method of internal standards and calibrationcurves.

Example 6 Preparation of Recombinant Tauons

Recombinant truncated forms of tau protein were prepared using “Erase aBase System” (Promega) according to the technical manual. The system isbased on exonuclease III specific digestion of inserted DNA startingfrom 5′ overhang. The rate of digestion was uniform at constanttemperature. The tau gene was cloned into pET17b vector throughNdeI-EcoRI restriction sites producing pET/T40. To the C-end of the genewas added KpnI restriction site and three stop codons in all threereading frames downstream of the KpnI site. The EcoRI enzyme leaves5′ends overhanging, the substrate for exoIII. KpnI leaves 3′ endsoverhanging which are resistant to exoIII digestion. 1 μg of pET/T40vector double digested with EcoRI, KpnI/NEB and ethanol precipitatedwere diluted in 20 μl 1×ExoIII buffer and digested by 80 u exoIII at 37°C./dig. rate 450 bases/minute. 2.5 μl samples were transferred afterExoIII addition at 30 s intervals into 7.5 μl S1-nuclease mix/1.5 uS1-nuclease for 1 sample on ice. The collected samples were incubated atroom temperature for 30 min to remove remaining single stranded tails.Klenow DNA polymerase was used to make blunt ends. DH5alfa competentcells were transformed directly with ligation mixtures of the samples.The subclones were screened by PstI-XhoI restriction and appropriateconstructs were sequenced using T7-primer in pET vector.

Expression, Purification and Quantification of Recombinant Tauons

Tauons were expressed in E. coli BL21 (DE3) (Studier, 1986). Singlebacterial colonies were inoculated to 500 ml of LB AMP (LB medium, 100μg/ml ampicillin). Bacterial cultures were grown at 37° C. on rotaryshaker until their OD₆₀₀ reaches 0.6-0.8 and then induced by adding IPTG(0.4 mM final concentration). After 3 hrs the bacterial cells werepelleted by centrifugation at 5000 g for 15 min at 4° C. (Sigma 6K15,rotor 12 500), and pellet of the cells quickly frozen in liquid nitrogenand stored at −70° C. until further use. For cell lysate preparation,bacterial pellet was resuspended in buffer A: (20 mM PIPES pH 6.9, 50 mMNaCl, 1 mM EGTA, 1 mM MgSO₄, 2 mM DTT, 0.1 mM PMSF), the cells weredisrupted by sonication on ice for 6 min and cell debris removed bycentrifugation at 45 000 rpm, 15 min at +2° C. (rotor TLA-12O.2,Beckmann Optima TLX). Supernatants were filtered through 0.22 μm filters(Millipore) and tauons were immediately purified by ion exchangechromatography on phosphocellulose (cellulose phosphate Whatman P11)column. After loading the sample the column was washed with 10 bedvolumes of the buffer A. Tauons were eluted with 20 ml of a lineargradient of NaCl (50 mM-0.5 M) in the buffer A. The 1 ml fractions werecollected and those containing proteins were identified on SDS-PAGE.Fraction containing tauons were pooled and dialysed against PBS 3×60 minat 4° C. Aliquots from the dialysate were vacuum dried (SpeedVac) andstored at −20° C. Recombinant tauons were quantified by PAGE, usingserially diluted bovine serum albumine (BSA) as mass standard markers.The gel was stained by Coomassie blue, dried and the intensity of BSAand tau bands was calculated using Scion Image (Beta 3b, Scion Corp.).The calibration curve for BSA was constructed and used forquantification of tauons.

Example 7 Isolation of Tauons from Human AD-Brain

For isolation of tauons from human AD brains a new approach partiallybased on the methods described by Kopke et al., (1993) and Greenberg andDavies (1990) was developed. Human brains, showing changescharacteristic for I.-III. Braak's stage of AD with short post mortemdelay (PMD) were selected. Blocks of the temporal lobe including theenthorinal and transenthorinal regions, amygdala and hippocampal regionwere selected. The tissue was dissected and immediately immersed intominimal essential medium (Gibco). Tissue was finely minced and pushedthrough a 150 μm mesh wire screen. At this stage the brain samples weredivided into two aliquots: sample A and sample B.

Sample A was further processed in 20 mM TRIS, pH 8, 0.32 M sucrose, 10mM β-mercaptoethanol, 5 mM EGTA, 1 mM EDTA, 5 mM MgSO₄, 5 mMbenzamidine, 10 mM glycerolphosphate, 6 mM phenylmethylsulfonylfluoride, 50 mM sodium fluoride, 5 μg/ml leupeptin, 1.5 μg/ml pepstatinand 2 μg/ml of aprotinine and centrifuged at 25 000×g for 35 min at 4°C. to remove cellular debris. The supernatant was then pelleted at 200000×g for 40 min. The resulting pellet was extracted with 8 M urea atroom temperature for 70 min and spun at 300 000×g for 45 min at roomtemperature. The supernatant was dialysed for 24 hrs against the 10 mMTRIS pH 7.6 with frequent changes and then dialysed for 24 hrs against100 mM MES, 0.5 mM MgCl₂, 1 mM EDTA, 2 mM EGTA, 1 mM dithiotreithol,0.75 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride and 50 mM NaF, pH2.7. The precipitated proteins were removed by centrifugation at 200000×g for 40 min. The 200 000×g supernatant was dialyzed against 25 mMMES, pH 6.4, 0.5 mM MgCl₂, 0.1 mM EDTA and 1 mM dithiotreithol andsubsequently fractionated on Cellulose Phosphate column which wasequilibrated with the same buffer. The column was loaded with 2 mg ofproteins and eluted with a linear gradient of NaCl (0-1M) inequilibrating buffer. The proteins eluted with 0.1-0.8 M NaCl wereevaluated by Western blotting and concentrated by speed vacuumapparatus.

Sample B was put into 10 volumes of cold buffer (10 mM TRIS, 1 mM EGTA,0.8 M NaCl, 10% sucrose, pH 7.4) in a glass homogenizer. Aftercentrifugation at 27 000×g for 30 min at 4° C., the supernatant wassaved and the pellet was homogenized with the buffer and centrifuged at27 000×g for 30 min. The 27 000×g supernatants from both centrifugationswere combined, adjusted to 1% (wt/vol) N-lauroylsarcosine and 1%(vol/vol) β-mercaptoethanol and incubated at 37° C. for 3 hrs whileshaking on shaker. After centrifugation at 35 000 rpm for 30 min, thepellet was homogenized in 5 ml of homogenizing buffer supplemented with1% mercaptoethanol and filtered through 0.45 μm filter. The filtrate wascentrifuged at 35 000 rpm for 1 hr. The pellet was resuspended in 50 mMTris, pH 6.8 and extracted with 2.5% formic acid for 2 min and thencentrifuged at 10 000×g for 10 min to pellet insoluble material. Thesupernatant was dialysed overnight at 4° C. against 10 mM Tris, pH 7.4and centrifuged as previous. The resulting supernatant was concentratedusing speed vacuum apparatus and evaluated by SDS-PAGE followed byWestern blotting.

Example 8 Purification of Normal Tau from Human, Pig and Cow BrainTissues

Tau was purified by the modification of the method of Lindwall andCole., 1984. Brain tissue was homogenizated (1 mg/ml) in 0.1 mM MES, 0.5mM MgCl₂, 1 mM EGTA, 1 M NaCl pH 6.5 and centrifuged at 100 000×g at 4°C. for 90 min. The supernatant was made up to 0.5% (v/v)2-mercaptoethanol, heated at 100° C. for 5 min and centrifuged at 20000×g at 4° C. for 30 min. This second supernatant was brought to 45%saturation in (NH₄)₂SO₄ and centrifuged at 20 000×g as above and theresulting pellet was resuspended in MES buffer without NaCl. Afterprecipitation with 2.5% (v/v) perchloric acid and a furthercentrifugation at 20 000×g the final supernatant was dialysed against 5mM Tris, pH 7.4 overnight at 4° C.

Example 9 Sequestration and Aggregation of Normal Tau into Tangles ofFilaments by Tauons

Increasing amounts of normal tau (5-100 μg/1001) were mixed with fixedamount of tauons isolated from fraction I (10 μg/100 μl). The reactionwas performed in a final volume of 100 ml of binding buffer (100 mM MESpH 7.6 containing 2 mM EGTA, 2% bovine serum albumin, 0.5 mM MgCl₂, 1 μMaprotinin and 20 μM leupeptin). The mixture was allowed to interact for45 min at room temperature, then overlaid on 150 μl of 80% sucrose inthe binding buffer and centrifuged for 1 hr at 100 000×g. The top 150 μlwas removed and the remainder was sonicated for determining ofinteraction between tauons and normal tau by radioimmuno-dot-blot assay.The presence of tau in the sucrose layer indicates sequest ration ofhealthy tau by tauons.

Example 10 Inhibition of Neuron Degeneration by Family of DC-11Monoclonal Antibodies

Neuronal blastoma cells and growth factor are plated on Petri dishes intriplicate. The first group received tauons only and the second onereceived tauons and the mixture of DC-11 monoclonal antibodies.

Detection of the Transfected Tauons by Immunofluorescence

Cells were permeabilized for 5 min at room temperature in 0.2% Triton X100 containing 80 mM PIPES, 1 mM MgCl₂, 1 mM EGTA, pH 6.6. Fixation ofthe cells was performed in 2% paraformaldehyde in the same buffer for 15min on ice. Tauons were detected by indirect immunofluorescence avidinrhodamine detection system (Sigma).

Detection of Early Inhibition of Neuronal Differentiation

The cells were grown with the differentiation inducing factors. Level ofdifferentiation was evaluated. The group of the cells harbouring tauonswithout antibodies had significantly impaired capacity to differentiate.However, group treated with mixture of tauons and antibodiesdifferentiated to comparable level with the cells from control grouptreated with irrelevant protein.

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1. An isolated antibody that: (a) specifically recognizes an epitope inthe amino acid sequence of SEQ ID NO: 1 of a truncated form of human tauprotein that comprises the amino acid sequence of SEQ ID NO: 1 andconsists of between 100 to 400 amino acid residues of the human tauprotein, the truncated form of the human tau protein beingconformationally different from normal human tau protein; and (b) doesnot specifically recognize normal human tau protein.
 2. The antibody ofclaim 1, wherein the truncated form of human tau protein is truncated atthe N-terminus.
 3. The antibody of claim 1, wherein the truncated formof human tau protein is truncated at the C-terminus.
 4. The antibody ofclaim 1, wherein the truncated form of human tau protein is truncated atboth termini.
 5. A hybridoma cell line producing an antibody that: (a)specifically recognizes an epitope in the amino acid sequence of SEQ IDNO: 1 of a truncated form of human tau protein that comprises the aminoacid sequence of SEQ ID NO: 1 and consists of between 100 to 400 aminoacid residues of the human tau protein, the truncated form of the humantau protein being conformationally different from normal human tauprotein; and (b) does not specifically recognize normal human tauprotein.
 6. A method for detecting truncated forms of human tau proteincomprising: (a) mixing a sample of brain tissue or cerebral spinal fluidwith an antibody that: (i) specifically recognizes an epitope in theamino acid sequence of SEQ ID NO: 1 of a truncated form of human tauprotein that comprises the amino acid sequence of SEQ ID NO: 1 andconsists of between 100 to 400 amino acid residues of the human tauprotein, the truncated form of the human tau protein beingconformationally different from normal human tau protein; and (ii) doesnot specifically recognize normal human tau protein; and (b) detecting abinding event between said antibody and the truncated form of human tauprotein, wherein detecting the binding event detects the truncated formsof human tau protein in the sample.
 7. The method of claim 6, whereindetecting the binding event comprises using a secondary antibody.
 8. Themethod of claim 7, wherein the secondary antibody is labeled.
 9. Themethod of claim 6, further comprising quantifying the amount of thetruncated form of human tau protein bound to the antibody.
 10. Themethod of claim 9, wherein quantifying the truncated forms of human tauprotein comprises using a standard preparation of the truncated form ofhuman tau protein.
 11. A method of inhibiting neuron degenerationcomprising administering to a neuron an antibody that: (i) specificallyrecognizes an epitope in the amino acid sequence of SEQ ID NO: 1 of atruncated form of human tau protein that comprises the amino acidsequence of SEQ ID NO: 1 and consists of between 100 to 400 amino acidresidues of the human tau protein, the truncated form of the human tauprotein being conformationally different from normal human tau protein;and (ii) does not specifically recognize normal human tau protein,wherein degeneration of the neuron degeneration is inhibited.
 12. Themethod of claim 11, wherein the antibody is an isolated DC-11 antibodyas produced by hybridoma cell line DC-11 (ECACC Deposit No: 00082215).13. The method of claim 11, wherein the antibody is an isolated DC-11/Iantibody as produced by hybridoma cell line DC-11/I (ECACC Deposit No:00082216).